Sarcosine oxidase (EC 1.5.3.1) is used as an enzyme for measuring creatine and creatinine in the body fluid, which are clinical indicators of diagnosis of muscular diseases and renal diseases, together with the other enzymes such as creatininase, creatinase and peroxidase. Sarcosine oxidase acts on sarcosine which is a substrate in the presence of water and oxygen to produce glycine, formaldehyde and hydrogen peroxide.
It has been known that such sarcosine oxidase is produced by bacteria belonging to genera Bacillus (JP-54-52789-A, JP-61-162174-A), Corynebacterium (J. Biochem., 89:599, 1981), Cylindrocarpon (JP-56-92790-A), Pseudomonas (JP-60-43379-A), and Arthrobacter (JP-2-265478-A). Technology to produce sarcosine oxidase on a large scale using a host such as Escherichia coli with a sarcosine oxidase gene obtained from these bacteria by a gene engineering technique has been also reported (JP-5-115281-A, JP-6-113840-A, JP-8-238087-A).
Along with recent liquefied reagents for clinical diagnosis, various stabilization methods of reagent components in a liquid have been investigated, and also for sarcosine oxidase used for reagents for measuring creatinine and creatine, one which is excellent in stability in the liquid has been desired. Our group previously reported a mutant type of sarcosine oxidase whose stability for metal ions was improved by modifying a wild type of sarcosine oxidase in a protein engineering manner (see e.g., JP-7-163341), but concerning long term storage stability in a diagnostic reagent, more improvement has been anticipated.
Furthermore, it has been known that conventional sarcosine oxidase also acts on proline which is one amino acid present in blood, and it has been pointed out that this can cause a true or false difference upon measuring creatinine and creatine (Rinsho Kagaku, 20:144–152, 1991; Seibutsu Shiryo Bunseki, 17:332–337, 1994). In order to solve this problem, our group reported sarcosine oxidase having a lowered action on proline by modifying the wild type of sarcosine oxidase in the protein engineering manner (JP-10-248572-A), but the action thereof on sarcosine which is an original substrate has been unclear, and more improvement has been desired.
It is an object of the present invention to provide modified sarcosine oxidase having an improved stability in a liquid.
It is another object of the present invention to provide sarcosine oxidase having a low reactivity to proline and having an excellent substrate specificity.
It is another object of the present invention to provide sarcosine oxidase having a lowered action on proline.